Interferons induce broad pleiotropic effects including growth inhibition, antitumor activity, hematopoietic regulation, control of cellular/humoral immune responses, and activity against viral and parasitic infection, accounting for interest in these cytokines as potential therapeutic agents. The induction of the ISG15 gene to generate the 17.1 kDa polypeptide ISG15 is one of the earliest responses to Type 1 interferons. ISG15 (formerly termed UCRP, the Ubiquitin Cross Reactive Protein) is the archetype of a recently discovered class of ubiquitin-like proteins that appear to regulate cellular function rather than to target susceptible proteins for degradation by the 26S proteasome. In contrast to the other ubiquitin-like proteins, ISG15 is not present in lower eukaryotes and thus represents a relatively recent evolutionary divergence. The biological effects of ISG15 are exerted through its covalent ligation to a small population of cellular targets that is distinct from those targeted by other ubiquitin-like proteins. Two-hybrid screens have recently identified a small class of highly conserved late interferon-induced proteins collectively termed the 1-8 family. The ca 14 kDa polypeptides bear remarkable yet cryptic sequence similarity to the conserved catalytic domain of ubiquitin conjugating enzymes (Ubc). The present proposal comprises three specific aims. Specific Aim 1 will test the hypothesis that 1-8 isoforms represent ISG15-specific conjugating enzymes through in vitro assays and stable cell transfections of active and dominant mutant forms of the polypeptides. Other studies will concentrate on functional differences among the different isoforms with respect to potential substrate specificity and cellular localization. Specific Aim 2 will focus on the 9-27/Leu13 isoform and its role in transducing antiproliferative and homotypic adhesion signals. Other studies will map protein-protein interaction motifs between 9-27 and the ubiquitous tetraspanin CD81 and their functional interactions with CD19-CD21. Specific Aim 3 will exploit recent advances in proteomics and mass spectroscopic protein fingerprinting to identify time-dependent changes in populations of targets for ISG15 conjugation in quiescent and interferon induced states. These studies are the first concerted functional survey of ISG15 conjugation and the enzymology of this modification.